Fusion between membranes is a ubiquitous yet poorly understood cellular process. Infection of cells by enveloped viruses is initiated by formation of a fusion pore between the virus and a host cell membrane. The process is induced by fusion proteins, and for many unrelated viruses these proteins share the common feature of three monomers that fold into six-helix bundles in their final structure. Among these proteins are the hemagglutinin (HA) of influenza virus and Env of HIV-1. Agents that inhibit bundle formation in HIV-1 Env prevent fusion and have been shown to have the potential to control the progression of AIDS. Vaccines against influenza are usually directed against HA. The formation of the six-helix bundle causes the transmembrane domains (TMDs) in the viral membrane and fusion peptides of the protein inserted in the target membrane to come into close proximity. A central question of the field is whether the movement of fusion peptides and TMDs toward each other is the direct cause of fusion pore formation. This will be tested for both HA and HI-1 Env using several approaches. For HA, the N-cap region and the region proximal to the TMD interact to cause proximity of the TMD and fusion peptide, so these regions will be mutated and the prediction that hemifusion, but not full fusion, occurs will be tested. The TMDs of HA must separate from each other to create proximity with the fusion peptides; preventing this separation by cross-linking monomers will determine which steps of fusion can occur without proximity. For Env, residues will be mutated to reduce bundle formation and the effects on fusion will be measured. Inhibitory peptides and mutation experiments will determine whether bundle formation drives fusion pore enlargement for Env. Recombinants of the bundle-forming portion of Env inhibit fusion; determining how they do so will either provide the free energy released by bundle formation or yield a means to explore associations between Env's. Extent of association between proteins will also be investigated by measuring kinetics of fusion as a function of HA density. The accumulation of HA-GFP into the region of contact and/or into large complexes, as fusion proceeds, will be explored. Protein associations will also be probed through the use of a simplified model system for Env receptor and coreceptor. These aims will be investigated by video fluorescence dye spread measurements, electrical admittance techniques, and laser scanning confocal microscopy, as appropriate.